DNA polymerases replicate the genomes of living organisms. For a review of polymerases, see, e.g., Hübscher et al. (2002) EUKARYOTIC DNA POLYMERASES Annual Review of Biochemistry Vol. 71: 133-163; Alba (2001) “Protein Family Review: Replicative DNA Polymerases” Genome Biology 2(1): reviews 3002.1-3002.4; and Steitz (1999) “DNA polymerases: structural diversity and common mechanisms” J Biol Chem 274:17395-17398.
In addition to a central role in biology, DNA polymerases are also ubiquitous tools of biotechnology. They are widely used, e.g., for reverse transcription, amplification, labeling, and sequencing, all of which are central technologies for a variety of applications, such as sequencing, nucleic acid amplification (e.g., PCR), cloning, protein engineering, diagnostics, molecular medicine and many others. The formats in which DNA polymerases are used vary widely, but generally the polymerase is present in solution when it is active. Useful formats include nucleic acid amplification and/or sequencing in automated amplification and/or sequencing devices, microtiter plates, microfluidic devices, PCR machines, and many others.
Active DNA polymerases captured to a solid phase, e.g., for use in solid phase DNA amplification, have not generally been available. Indeed, surface immobilization has been shown to disrupt DNA polymerase activity, in at least some settings. For example, affinity immobilization of Taq DNA polymerase has been used to suppress DNA polymerase activity until the polymerase is released from affinity capture (by application of high temperature). See, e.g., Nilsson et al. (1997) “Heat-Mediated Activation of Affinity-Immobilized Taq DNA Polymerase” BioTechniques 22:744-751.
An RNA-polymerase system in which an active RNA polymerase is coupled to a surface through an anti-HA antibody binding to an HA-tagged polymerase is described by Adelman et al. (2002) “Single Molecule Analysis of RNA Polymerase Elongation Reveals Uniform Kinetic Behavior” PNAS 99(21):13538-13543. This RNA polymerase was labeled at the N-terminus with a His-6 tag (for purification of the enzyme prior to attachment) and a C-terminal HA tag for binding to a surface. The anti-HA antibody was non-specifically adsorbed on the surface, which was additionally blocked with milk protein to reduce non-specific binding. The assay that this system was applied to is rather specialized and complex, involving detecting the physical forces exerted by an RNA polymerase during transcription. Applicability of this RNA polymerase system to other enzymes and to general topics such as detecting amplification products in general for high-throughput sequencing, labeling and amplification methods is unclear. For example, RNA polymerase is much slower than many DNA polymerase (5 bp/s vs. about 750 bp/s maximum in vitro), and RNA pols cannot easily be used for DNA amplification, SNP detection or the like.
While there are many solution phase applications of DNA polymerases that are useful, further applications in which the polymerase is active on a solid surface would be desirable. For example, the ability to fix the polymerase to a selected location on a surface could permit the creation of arrays of DNA polymerases, the activity of which could be detected using available array reader technologies. Furthermore, the ability to position a polymerase on a surface could permit single-molecule analysis of DNA products at the site on the surface where the DNA polymerase resides. This, in turn, would permit real-time monitoring of polymerase reactions, e.g., as in real time PCR (RT-PCR) or real-time sequencing. The present invention provides these and other features that will be apparent upon review of the following.